The structure of the Rep endonuclease domain (Hickman et al., 2002) revealed that it is unrelated to all other structurally characterized nucleases and provided the first view of an HUH superfamily member. Rep is homologous to the origin binding domains of the SV40 T antigen (Luo et al., 1996) and replication initiation protein E1 of bovine papillomavirus (Enemark et al., 2000). The HUH residues, which bind the catalytically required metal ion, converge with a helix bearing the two active site tyrosine residues to create the enzyme active site cleft. Subsequent co-crystal structures of the Rep nuclease domain with oligonucleotides representing two specific regions of the AAV genome showed that the nuclease domain uses two different protein surfaces to recognize its DNA target. One surface binds a hairpin that is at the very tip of the viral genome and the other recognizes a repeated tetranucleotide sequence close to the genome ends that constitutes the Rep binding site. These structures allowed us to propose a model for the assembly of a hexameric Rep-DNA complex that is poised to nick the viral DNA and begin unwinding it as a prelude to replication. It seems likely that site-specific integration also begins with a nick at a related sequence in human chromosome 19.[unreadable] Although it is believed that Rep assembles as a hexameric helicase, such assemblies had not previously been observed and the mode of Rep multimerization remains controversal. We have determined the conditions necessary to assemble a hexameric form of full-length Rep on AAV DNA sequences, and have used binding assays and DNA footprinting methods to determine the limits of the Rep binding site on AAV viral DNA. We can generate hexameric complexes of Rep on both single-stranded and dsDNA substrates which have a 3' single-strand extension, and crystallization trials are underway.[unreadable] [unreadable] Enemark, E.J., Chen, G., Vaughn, D.E., Stenlund, A., and Joshua-Tor, L. (2000) Mol. Cell 6, 149-158.[unreadable] Flotte, T.R. (2005) Pediatric Res. 58, 1143-1147.[unreadable] Hickman, A.B., Ronning, D.R., Kotin, R.M., and Dyda, F. (2002) Mol. Cell 10, 327-337.[unreadable] Im, D.S. and Muzyczka, N. (1990) Cell 61, 447-457.[unreadable] Le Bec, C. and Douar, A.M. (2006) Gene Ther. 13, 805-813.[unreadable] Lee, H.C., Kim, S.J., Kim, K.S., Shin, H.CV., and Yoon, J. W. (2000) Nature 408, 483-488.[unreadable] Luo, X., Sanford, D.G., Bullock, P.A., and Bachovchin, W.W. (1996) Nat. Struct. Biol. 3, 1034-1039.